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Transfections
Lab protocols for various ways of transfecting phoenix or 293T cells Lipofectamine transfection of phoenix cells or 293T cells *Change media on phnx cells right before procedure (proliferative burst helps cells take up lipo) *Lipofectamine in 4C, bring to RT *Optimem (or DMEM w/o FBS) in TC 4C, bring to RT *6well format: 500uL mix of lipofectamin and dna (total volume) **Always 3uL lipo: 1ug DNA **Scale up: 10cm: 2mL total mixture (1mL lipo+opto, 1mL opto+DNA); 15cm: 4mL mix *We’ll be infecting 2ug (in 6 well format), so use 6uL lipo *(# reactions + 0.5) *250uL = final volume of lipo master mix *(# reactions + 0.5)* ug DNA/rxn *3 = uL Lipo in master mix *Put Optimem in conical or eppendorf *Let lipo come to RT and vortex, then add to optimem (otherwise will stick to plastic) *Let master mix sit 5 mins *Mix 1:1 with DNA (2ug) cued to 250uL with optimem *Let reaction mixtures sit 20-40mins RT *Pipette onto cells *Leave on overnight, but no more than 18hours (sit at least 8 hours) *Change media before 18hrs *Continue making virus or splitting cells, whatever your experiment requires Notes: *Lipo infection of 293Ts is exactly the same except requires inclusion of packaging constructs (8.91 and pUC-MDG) **8.91= lentiviral gag, pol **pUC-MDG= VSVG-pseudotyped viral envelop *Fugene protocol is practically the same. Fugene transfections Exactly the same as with lipofectamine. Just use FuGene instead. Retrovirus Protocol from VLP Virus production: *Day 1 - Plate 900K phoenix cells per well of 6 well plate. (want cells ~40-50% confluent next day) *Day 2 - Transfect 2 ug retroviral plasmid per well using FuGene. *Use 3:1 FuGene to DNA mix: 6ul Fugene in 94ul serum-free DMEM—incubate at rt 5min * Add 2ug DNA to Fugene mix—incubate at rt for 15min *Add mixture to cells (no need to change media) *Day 3 - Change media on transfected phoenix cells (add 3 mL media per well) and put at 32 degrees for virus collection. *Day 4-6 - Collect virus every 24 hours. Add back 3 mL media. Filter virus through 0.45 um filter and freeze on dry ice prior to storage at -80. Virus can also be used fresh. (Make sure you bleach anything that touches virus.) Infection: *Day 1 - Plate 30K keratinocytes per well of 6-well plate. *Day 2 - Infect keratinocytes with 2-3 mL virus (whatever you harvest from 1 well during virus production). Add polybrene (located in same -20 freezer as FBS) to final concentration of 1ug/mL - stock is at 1 mg/ml so use at 1:1000. Place plate in centrifuge in plate spinners. Spin at 32 degrees, 1100-1200 rpm for 1 hr. Wash cells 2x PBS. Add back 50:50 media. (Make sure you bleach anything that touches virus.) *Day 3 - Infect keratinocytes with 2nd round of virus (same as above). *Day 4 - Transfer infected cells to 6 cm plate (add puro selection to 1ug/mL - 1:1000 of stock located in same -20 freezer). You can expand this population and check for knockdown at 3 day after selection by western blot. Calcium Phosphate transfections